Analytical methods that combine the selectivity of enzymes with the sensitivity of amperometric detection are of interest to the diagnostic industry. The reduction of the nicotinamide co-enzymes (NAD and NADP) is particularly important because they are produced in reactions catalyzed by dehydrogenases. Dehydrogenase catalyzed reactions according to the equation: ##STR1## play an important role in biological cells and analytical reactions. Several hundred different dehydrogenases are known which selectively catalyze the conversion of different substrates into products. When the substrate is oxidized, the coenzymes NAD.sup.+ and NADP.sup.+ are reduced to NADH and NADPH respectively. These co-enzymes are a necessary element in the reaction due to their ability to act with the enzymes to form an energy-transferring redox couple.
The co-enzymes NAD.sup.+ and NADP.sup.+ are expensive chemicals making their regeneration by reoxidation to their original state imperative if they are to be economically used in low cost, disposable, analytical devices. The NADH is oxidized directly at different base electrode materials only with high overvoltages on the order of 1 volt. However, a decrease in this over-voltage can be obtained by the immobilization of functionalities on the electrode surface which mediate the electron transfer from NADH to the electrode. Such mediators are typically selected from materials which may be reoxidized electrochemically without excessive overvoltages rendering them useful as an auxiliary system for electrochemical regeneration. Various mediator compounds suitable for this purpose are known. In U.S. Pat. No. 4,490,464 there are mentioned, by way of background, mediators such as phenazine methosulphate (PMS); phenazine ethosulphate (PES); thionine and 1,2-benzoquinone. This patent goes on to describe electrodes which are modified to catalyze the oxidation of NADH, NADPH or analogs thereof by imparting to the electrode surface as mediator a condensed aromatic ring system comprising at least three and preferably four or more condensed aromatic rings with or without heteroatoms. More particularly, this reference describes the electron exchange with the co-enzyme or analog thereof by structural elements comprising one of either alkyl phenazinium ions, phenazinium ions, phenazinones, phenoxazinium ions, phenoxazinones, phenothiazinium ions or phenothiazinones.
Fulty et al provide a literature review of various mediators, which they describe as "electron shuttles", which provide redox coupling between the electrode and the redox center of the co-enzyme in Analytical Chimica Acta. 140 (1982) Pp. 1-18. Perhaps the best known mediators for use on graphite electrodes are phenothiazinium and phenoxazinium salts such as Meldola's Blue (II): ##STR2##
In J. Electroanal. Chem., 287 (1990) Pp. 61-80, there is described a chemically modified graphite electrode for oxidation of reduced NAD based on the phenothiazine derivative 3-.beta.-naphthoyl-toluidine blue O (III): ##STR3##
Persson et al have published a comparative study of some 3,7-diaminophenoxazine derivatives for the electrocatalytic oxidation of NADH in J. Electroanal Chem., 292 (1990) 115-138.
In U.S. Pat. 4,810,636, there are described 7-hydroxy-9H-acridin-2-one chromagens which have been derivatized at the 7-hydroxy position with an enzymatically cleavable group and substituted at the 9-position with alkyl or aryl groups.